Method, system and computer program product for technical management and biocontrol of disease in animal production systems

ABSTRACT

A system, method and computer program product for controlling disease at an end user location, including (a) testing a range of candidates including at least one of: (i)  Bacillus  species, (i)  Bacillus  strains, (iii) species of beneficial bacteria (iv) strains of beneficial bacteria and (v) strains of beneficial bacterial viruses, against samples including at least one of pathogenic  Vibrio , Gram negative pathogenic bacteria and Gram positive pathogenic bacteria taken from an end user location; (b) performing at least one of the following steps: (i) selecting one or more of the candidates that one of inhibit and attack at least one of the samples by direct inhibition of at least one of in situ antibiotic production, competitive exclusion, production of enzymes that degrade quorum sensing molecules, and (ii) testing a range of quorum sensing inhibitor compounds against the samples; and (c) performing the steps (a) and (b) for the end user location, including one of a country, major region and individual end user location, to target microbial technology to use in bio-control of disease specific to the end user location.

CROSS REFERENCE TO RELATED CASES

The present invention claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 60/0234,784 ofVillamar et al., entitled “METHOD, SYSTEM AND COMPUTER PROGRAM PRODUCTFOR TECHNICAL MANAGEMENT AND BIOCONTROL OF DISEASE OF AQUATIC ANIMALS,”filed on Sep. 25, 2000 and is related to U.S. Provisional PatentApplication Ser. No. 60/213,538 of Villamar et al, entitled “BIOACTIVEFOOD COMPLEX METHOD FOR MAKING BIOACTIVE FOOD COMPLEX PRODUCT AND METHODFOR CONTROLLING DISEASE,” filed on Jun. 23, 2000 and PCT ApplicationSer. No. PCT/US/16489 of Villamar et al, entitled “BIOACTIVE FOODCOMPLEX, METHOD FOR MAKING BIOACTIVE FOOD COMPLEX PRODUCT AND METHOD FORCONTROLLING DISEASE,” filed on 22 Jun. 2001, the entire contents of allof which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the field of biocontrol ofdisease in animal production systems and more specifically to a method,system and computer program product for the technical management andbiocontrol of disease in animal production systems by use of microbialbiotechnology. The present invention includes use of varioustechnologies described in the references identified in the appended LISTOF REFERENCES and/or cross-referenced throughout the specification bynumerals in brackets corresponding to the respective references, theentire contents of all of which are incorporated herein by reference.

2. Discussion of the Background

Aquatic farming is the fastest growing sector of internationalagribusiness today (estimated farm gate value over US$30 billion/yr),with double-digit growth in major geographic regions where fish andshrimp are grown commercially. Sustained growth in aquaculture is neededto compensate for declining traditional fisheries and to meet a growingdemand for high-value protein. Harvests of marine and freshwater finfishand shellfish for human consumption (herein defined as seafood) from theworld's capture fisheries has been relatively stagnant at about 60million tonne/yr over the last several years. During this time, majorcommercial fishing grounds have been classified as “Fully Exploited” or“Over Exploited.” The Food and Agriculture Association (FAO) estimatesworld annual demand for seafood in the year 2010 at 110 to 120 milliontonne. Best-case projections show a world supply of about 114 milliontonne with less-favorable estimates at about 74 million tonne. Arealistic scenario shows a deficit of 36 million to 46 million tonne.[1] Based on FAO and other estimates, about 10 million tonne of aquafeedwill be used in the world in 1999 to feed the cultured of fish andshrimp.

At the same time, all segments of the aquaculture industry, e.g.,shrimp, finfish, shellfish, etc., are being strongly affected bydisease, especially bacterial diseases. Farmed shrimp production, whichreached 737,200 tonne live weight in 1998 with an estimated trade valueof about US$6 billion, is the most valuable export aquaculture crop andthe hardest hit by disease in terms of financial losses to the industry.About 80% of the world's shrimp production takes place in Asia, the restpredominantly in Latin America. Thailand has been the world's largestfarmed shrimp producer with over 210 000 tonne/yr and Ecuador was secondwith over 130,000 tonne; the production in both countries have beensignificantly affected by disease.

The damage caused by bacterial and viral diseases to the shrimp industryhas been in the billions of dollars. Pathogenic Vibrio bacteria andviruses, such as Yellow Head Virus, White Spot (WSV) and Taura Virushave been among the most damaging pathogens. Central America was hithard in 1999 by WSV and pathogenic Vibrio disease, causing great lossesin production, for example, up to 50% in some countries. WSV can resultin 100% mortality within the first few weeks after stocking shrimpponds. Ecuador, the largest shrimp producing country in the WesternHemisphere was severely affected by WSV in 1999 and is estimated to havelost more than 40% of its annual production with no signs of significantrecovery to date.

Chemicals and heavy use of antibiotics are the most commonly usedmethods to control shrimp diseases. However, these processes can beineffective and dangerous. Indiscriminate use of antibiotics anddisinfectants has led to an increase in bacteria having multipleantibiotic resistances. Many of the pathogens appear to have mutated tomore virulent forms than were present a decade ago, resulting in greaterrates of shrimp mortality. Thus, the incidence of the disease has beenexacerbated by the actions of the shrimp farmers themselves usingantibiotics.

Use of most chemicals and drugs is prohibited in the United Statesshrimp farming, wherein shrimp imports are tested for chemical residuesand U.S. authorities have rejected shipments. Furthermore, the concernof potential transfer of antibiotic resistance to human pathogens andthe negative marketing image created by use of chemicals and drugs canslow the growth and damage the aquaculture industry in the long-term.

The major disease agents around the world in aquaculture are bacteria,especially Vibrio species in marine systems. Often, a combination ofviral and bacterial disease appears to be a widespread cause ofmortality. For example, shrimp exposed to heavy environmental stress canbe severely weakened by Vibrio sp. favoring invasion and increasingpathogenicity by virus, such as WSV. Conversely, shrimp that areinfected by virus, but normally tolerate its pathogenicity can succumbto the virus with the additional stress of Vibrio infection.

Currently, pharmaceutical companies sell antibiotics to aquatic farmers.In addition, it is quite likely that problems have been exacerbated bythe use of other antimicrobial compounds. Chlorine is widely used inhatcheries and ponds, but its use stimulates the development of multipleantibiotic resistance genes in bacteria [28, 29]. If antibiotics areused to kill bacteria, there are always some bacteria that survive,either strains of the pathogen or others, because they carry genes forresistance. These will then grow rapidly because their competitors areremoved. Virulent pathogens that then re-enter the tank, perhaps fromwithin biofilms in water pipes or in the guts of animals, can thenexchange genetic information with the resistant bacteria and survivefurther doses of antibiotic. Thus, antibiotic-resistant strains ofpathogens evolve rapidly [35].

The transfer of resistance to human pathogens and gut bacteria is ofmajor concern. Such transfers probably happen easily and often. A genecoding for tetracycline resistance has been transferred betweenPrevotella, bacteria that normally live in the rumen of farm animals,and Bacteriodes, bacteria that normally live in human guts [30].Resistance plasmids (R plasmids) encoding for many antibiotic resistancegenes were transferred between pathogenic and non-pathogenic Gramnegative bacteria in several environments including sea water [31]. Inthe presence of tetracycline concentrations that were not high enough tokill the bacteria, the rate of gene transfer between Vibrio cholerae andAeromonas salmonicida increased 100 times.

This work raises questions not only about the use of antibiotics inaquaculture, but about the use of bacteria closely related to pathogenicspecies as probiotics. Not only antimicrobial resistance genes, but alsogenes for virulence can be transferred by R plasmids and transposons[32]. As the R plasmids can transfer genes between widely differentbacteria in the Gram negative group, it would be potentially dangerousto use Vibrio or Pseudomonas, for example, as probiotics. However, theuse of such bacteria is promoted, particularly Vibrio alginolyticus[33]. Based on observations by the present inventors, it is noted thatthe efficacy of Vibrio species as probiotics is short lived. Indeed,strains of Vibrio alginolyticus have been reported as virulent pathogensof shrimp larvae [34].

Throughout Asia, prawn farmers use antibiotics in large quantities.Warehouses supplying the industry in all the major centers sell a rangeof antibiotics in containers of 500 g or more in size. The antibioticsin current use include fluoroquinolones especially norfloxacin andenrofloxacin, furazolidone, oxolinic acid, oxytetracycline, trimethoprimand sulphadiazine. It is difficult to find out just how much antibioticuse there is in the industry, but it is possible to make an estimatefrom feed usage and production. In 1994, Thailand produced about 250,000tonnes (a quarter of the world production) of farmed prawns, whichconsumed about 500,000-600,000 tonnes of feed. With the diseaseproblems, prawn production has dropped to as low as 150,000 tonnes. Foreach crop at semi-intensive to intensive scales of production, farmersuse 5-10 g antibiotics per kg feed at least once per day at weeklyintervals; some use them for more extensive periods. Thus, asantibiotics would be used in about 10% of feed, the antibiotic usage inprawn farm production Thailand alone would be about 300-600 tonnes peryear. And this does not include that used in hatcheries for fryproduction. As much of this will end up producing bacteria with multipleantibiotic resistance in farm effluents that then contaminate coastalwaters, the potential impact on human health is significant [32, 35].

The overall strengths of antibiotics in the market place are long-termconditioning of individuals to the value of antibiotics for human andanimal therapy; strong and well-financed marketing, and immediateshort-term benefit. In most cases, the farmer does not see the long-termresistance build up and increased virulence until too late. Althoughsome aquaculture producers are now realizing that they must move awayfrom antibiotics, there are many who still use antibiotics. Antibioticsare recognized for their serious contribution to the collapse or declineof the shrimp industries in Taiwan, China, India, Thailand, andPhilippines.

The use of probiotics to fight disease can be much more effective thanuse of antibiotics. The term “probiotic” was coined in the 1970s as acontrast to antibiotic and refers to beneficial bacteria found in thestomachs and intestines of animals that aid the animal in digestion andin fighting hazardous, disease-causing bacteria That is, when beneficialbacteria that are normal internal residents of the animal are added inlarger numbers than present naturally, they promote health of theanimal, in other words, they help the animal fight disease organisms.The probiotic approach to fighting disease does not share theabove-noted disadvantages of an antibiotic approach. However, simplyadding beneficial bacteria to aquaculture systems typically does notnecessarily provide a solution to disease.

Among the companies attempting to combat disease in aquaculture arefermentation companies that manufacture or sell microbial products forindustries other than aquaculture. However, fermentation companiesand/or their marketers typically do not understand aquatic microbialecology and aquaculture. These companies typically sell products thatare inappropriate, ineffective and/or too expensive for aquatic farmers.

There are several dozen companies around the world selling bacteriareferred to as “probiotics,” although in most cases, the bacteria theysell do not meet the definition or function of probiotics, and indeedsome are pathogens. Many companies are now selling Lactobacillusbacteria for fighting disease in shrimp aquaculture, but Lactobacillusis a probiotic for terrestrial animals and is the wrong bacterium forseawater and crustaceans. In many cases the microbial products may besuitable for wastewater treatment and bioremediation, but are notappropriate for disease control in aquaculture i.e., the bacteria do nothave the genetic or molecular capacity to prevent disease. Mostcommercial products have very low concentrations of bacteria making themineffective when added to large water volumes in aquaculture containmentsystems such as grow-out ponds. Some commercial companies have attemptedto overcome the problem of low bacterial concentrations by requiringon-site fermentation by customers to generate sufficient numbers ofbacteria to add to the large quantities of water in aquaculturecontainment systems.

An exemplary background art system of FIG. 10 includes a technologydeployer 1002, a manufacturer 1004, a distributor 1006 and end users1008 to 1010. In FIG. 10, the technology deployer 1002 determinesbacteria 1038 that are perceived to have beneficial properties basedpresumably on scientific literature or based on their experience and ontheir ability to manufacture 1004 the bacteria 1038 in low concentrationform by commercial fermentation methods. The distributor 1006, and /orthe manufacturer 1004 in the case where sales are direct, then sellsthese bacteria 1038 to the end users 1008 to 1010. The more advancedtechnology deployers may perform bioassays in their labs to test theability of their bacterial strains, which they are able to producecommercially, to inhibit generic pathogens, but typically do not testagainst strains selected from individual end user 1008 to 1010locations. In some cases, the product 1038 must be further fermented inmedia by the end users 1008 to 1010 to increase the number of microbesbefore application at the end user 1008 to 1010 locations.

The above solutions are not effective in disease prevention and do notoffer biocontrol of disease in aquaculture systems due to: (i) poorunderstanding of the physical, chemical and biological factors thataffect disease control in commercial aquaculture systems, (ii) lack oftechnical support at the end user location (iii) lack of management ofinformation generated by end user and its timely linkage to deploymentof an appropriate technological response to epizootic conditions (iv)microbial products that do not prevent disease in commercial aquaculturesystems, (v) microbial products that are not cost effective, and (vi)microbial products that are not user-friendly.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is to provide a novel method,system and computer program product for deploying effective control ofdisease in aquatic and terrestrial animal production systems includingintegration of technical and economic factors of such animal productionsystems with application of appropriate microbial technology to manageand prevent disease in such animal production systems.

Another object of this invention is to provide a novel method, systemand computer program product for providing technology and know-how tohelp prevent or control production losses to White Spot Virus, Vibrioand other bacterial diseases affecting commercial shrimp farming.

Another object of this invention is to provide a novel method, systemand computer program product for commercial application of microbialbiotechnology and quorum sensing molecule inhibitors to help prevent orcontrol losses to diseases affecting commercial shrimp farming, finfishand mollusc industries and retail aquarium sectors.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, basedon collection of appropriate data at end-user locations, analysis of thecollected data, synthesis of biotechnology solutions and appropriateapplication programs and timely deployment of thereof.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, basedon data collected using biosensors, based on DNA probes targeting genesfor virulence in bacteria and genes for identifying typical pathogenicbacteria.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, whichare safe, non-regulated, environmentally friendly bacterial isolatesfrom nature.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, whichallow an aquatic farmer to employ effective bio-control of diseases andavoid use of harmful antibiotics and other drugs and chemicals, helpingto remove these from the human food chain.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, whichhelp make shrimp farming “green” and sustainable.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors formaintaining health of aquatic species, including food finfish, shellfish(oysters, clams) and ornamental species in the retail aquarium trade.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, whichare ecologically and fight diseases that affect health of aquaticspecies, including food finfish, shellfish (oysters, clams, abaloneetc.) and ornamental species in the retail aquarium trade.

Another object of this invention is to provide a novel method, systemand computer program product for providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, whichare carried “inside” an animal feed and are delivered via animal feeds,fertilizers and other products used in aquaculture.

Another object of this invention is to provide a novel method, systemand computer program product providing products containing highlyconcentrated bacteria and/or quorum sensing molecule inhibitors, whichare exported to distributors as concentrates, which are then blendedwith appropriate carriers for re-sale and/or direct farm use.

The above and other objects are achieved according to the presentinvention by providing a novel system, method and computer programproduct for controlling disease at an end user location, including (a)testing a range of candidates including at least one of: (i) Bacillusspecies, (i) Bacillus strains, (iii) species of beneficial bacteria (iv)strains of beneficial bacteria and (v) strains of beneficial bacterialviruses, against samples including at least one of pathogenic Vibrio,Gram negative pathogenic bacteria and Gram positive pathogenic bacteriataken from an end user location; (b) performing at least one of thefollowing steps: (i) selecting one or more of the candidates that one ofinhibit and attack at least one of the samples by direct inhibition ofat least one of in situ antibiotic production, competitive exclusion,production of enzymes that degrade quorum sensing molecules, and (ii)testing a range of quorum sensing inhibitor compounds against thesamples; and (c) performing the steps (a) and (b) for the end userlocation, including one of a country, major region and individual enduser location, to target microbial technology to use in bio-control ofdisease specific to the end user location.

In another aspect of the present invention there is provided a novelapparatus and method for delivering microbes in an aquarium, includingdelivering the microbes in the aquarium via a device; and configuringthe microbes to maintain a healthy microbial flora of the aquarium andof animals residing in the aquarium, to help prevent disease in theanimals residing in the aquarium and to help maintain the aquariumclean.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a top-level system diagram for control of disease at end userlocations, according to the present invention;

FIG. 2 is a diagram of products, processing and end user applications,according to the present invention;

FIG. 3 is a diagram of a product application to a commercial and/orhobby aquarium, according to the present invention;

FIG. 4 is another embodiment of the system of FIG. 1 further includingone or more downstream manufacturers, according to the presentinvention;

FIG. 5 is a diagram for illustrating bio-control of disease in water,according to the present invention;

FIG. 6 is a diagram for illustrating bio-control of disease in a gut ofan animal, according to the present invention;

FIG. 7 is a flow chart for illustrating the operation of the system ofFIG. 1, according to the present invention;

FIG. 8 is a detailed network system diagram of the system of FIG. 1,according to the present invention;

FIG. 9 is an exemplary computer system that may be programmed to performone or more of the processes of the present invention; and

FIG. 10 is a top-level Background Art system diagram.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention includes recognition that aquatic farmed animalsare surrounded by a milieu that supports opportunistic pathogensindependently of the host animal, and so the bacterial populations,including pathogens, can reach high abundance around the animal [3].Vibrio grow attached to algae, and may reach high population densitiesafter being ingested with the algae and then excreted with lysed algaein faecal pellets by zooplankton; they are gut bacteria in fish andprawns/shrimp as well as zooplankton [5]. In aquaculture ponds, whereanimal and algal population densities are very high, Vibrio numbers arealso high compared to the open sea. Where pathogen numbers are high thephenomenon of quorum sensing can come into play, and result in theactivation of virulence genes that would not be turned on whenpopulation density is low [27]. The onset of prawn disease due toexposure to high numbers of Vibrio , especially when pathogenicity hasincreased by overuse of antimicrobial compounds, indicates that adefense is needed.

The species composition of a microbial community, such as that in apond, will be determined partly by stochastic phenomena, that is,chance, and partly by physiological factors. In other words, there areboth predictable and unpredictable factors that allow one species togrow and divide more rapidly than others, and thus dominate numerically.Chance favors those organisms that happen to be in the right place atthe right time to respond to a sudden increase in nutrients, e.g. fromthe lysis of algal cells or the decomposition of feed pellets that fallaround them. The farmer can manipulate the species composition byseeding large numbers of desirable strains of bacteria or algae; inother words, by giving chance a helping hand [13, 14].

Competitive exclusion is one of the ecological processes that can bemanipulated to modify the species composition of a soil or water body orother microbial environment. Small changes in factors that affect growthor mortality rates will lead to changes in species dominance. We arestill a long way from knowing all the factors that control bacterialspecies growth rates and even the complete species composition innatural environments, but enough is known to argue that it is possibleto change species composition by making use of competitive exclusionprinciples [13, 14]. Thus bacteria can compete by secretingantimicrobial compounds that do not necessarily kill all theircompetitors, but increase mortality rates just enough to tip the balancein resource utilization. For example, if a Bacillus strain produces anantibiotic that inhibits a Vibrio , then the Vibrio 's mortality ratewould increase, shifting the dominance to the Bacillus , even if theantibiotic were not produced at high enough concentration to kill all ormost Vibrio cells directly.

Microbial ecology and biotechnologies have advanced in the last decade,to the point that commercial products and technologies are available fortreating large areas of water and land to enhance population densitiesof particular microbial species or biochemical activities. The practiceof bioremediation (or bioaugmentation) is applied in many areas, butsuccess varies greatly, depending on the nature of the products used andthe technical information available to the end user. The bacteria thatare added must be selected for specific functions that are amenable tobioremediation, and be added at a high enough population density, andunder the right environmental conditions, to achieve the desiredoutcomes [13]. Bioaugmentation and the use of probiotics are significantmanagement tools, but their efficacy depends on understanding the natureof competition between particular species or strains of bacteria. Theyrely on the same concepts that are used successfully for soilbioremediation and probiotic usage in the animal industry.

Probiotics such as the Gram positive Bacillus offer an alternative toantibiotic therapy for sustainable aquaculture. Bacillus species arecommonly found in marine sediments and therefore are naturally ingestedby animals such as prawns that feed in or on the sediment. There areseveral reasons why it is better to add Bacillus rather than antibioticsto control Vibrio species. Many different antibiotic compounds arenaturally produced by a range of Bacillus species. Other bacteria areunlikely to have resistance genes to all the antibiotics at one time,especially if they have not been exposed to the Bacillus previously.Bacillus secrete many enzymes that degrade slime and biofilms and allowBacillus and their antibiotics to penetrate slime layers around Gramnegative bacteria. Furthermore, Bacillus compete for nutrients and thusinhibit other bacteria from growing rapidly. Thus any resistant bacteriacannot multiply readily and transfer resistance genes. Bacillus alsocompete for space on surfaces (e.g., the gut wall, etc.) and displaceother bacteria and prevent their population density from becoming toohigh [12-15].

The composition of microbial communities in aquaculture ponds and tankscan be changed by adding selected probiotic bacteria that displace anddirectly inhibit deleterious bacteria and virus under appropriatephysico-chemical and biological conditions, thus affording biocontrol ofdiseases. Mortalities of shrimp and fish caused by pathogenic luminousVibrio species bacteria and virus can be controlled in this manner [12,15]. The addition of beneficial microbes to aquaculture systems can alsoimprove water quality by speeding up processes of waste degradation,nitrification and denitrification [8, 9]. Control of disease in shrimpand fish culture can be achieved by mixing selected microbial speciesdirectly into the water and into the feed of the animals.

Furthermore, manipulating physico-chemical factors can alter themicrobial community composition of water bodies, which is influenced bythe physico-chemical environment. Thus disease control is an integrationof adding selected microbes to animal containment systems such as pondsand to the feed, and adding various chemical compounds and changingphysical factors, such as water mixing rates.

Accordingly, microbial biotechnology can provide a solution to the mostserious problems in aquaculture and can be applied to every link of theaquaculture value chain: from use of direct-fed microbials in aquafeedsfor all life cycle stages (e.g., broodstock, larvae, juveniles, adults.etc.), to bio-augmentation of hatchery, nursery, grow-out and transportwater systems, and more.

It is important to recognize that management of microbial ecologythrough the application of biotechnology to aquaculture is not simply acure after the disease occurs, but a preventative process and that virusdiseases are not easy to control. Positive results can be achieved whenattention is paid to all facets of pond and hatchery operations. Thus,according to the present invention, successful management of White SpotVirus and other diseases requires establishment of best practices andtraining services to manage pond ecosystems, with particular emphasis onthe microbial community.

The use of probiotics in fish culture is less developed than in shrimpculture, but probiotics that control fungus (e.g., Saprolegnia) andbacterial gut disease (e.g., Aeromonas) in a high value fish (Latescalcarifer) have been found. Accordingly, the present invention extendsinto the fields of fish culture and other aquatic animals.

Thus, with the present invention, a great opportunity to capture marketleadership in biocontrol of disease through the application of microbialbiotechnology to manipulate microbial communities in the gut and waterand to create customer-specific solutions to disease is possible. Thepresent invention will allow this result to be achieved by: (i) applyingexpert understanding of the physical, chemical and biological factorsthat affect disease control in commercial aquaculture systems, (ii)providing technical support at the end user location and trainingaquatic farmers to manage the microbial ecology of the pond and of thegut of aquatic livestock through use of appropriate biotechnology, (iii)directing and using technical information generated by end user todeploy appropriate technological responses to prevent and manageepizootic conditions (iv) providing products containing inhibitors ofquorum sensing molecules in bacteria and/or microbial productsspecifically designed to prevent disease in commercial aquaculturesystems, (v) providing products containing inhibitors of quorum sensingmolecules in bacteria and/or highly concentrated microbial products thatare affordable by end users, (vi) providing products containinginhibitors of quorum sensing molecules in bacteria and/or microbialproducts that are very user-friendly and (vii) integrating one or moreof the items (i) to (vi) described above with the economic andproduction objectives of animal producers allowing effective biocontrolof disease where none exists today.

The deployment of the technology of the present invention is preceded byan evaluation of the production economics factors of the end user, forexample, including livestock survival rate, growth rate, feed conversionratio, biomass yield, costs of seedstock, costs of feed, costs offertilizer and other additives, costs of energy, costs of labor, costsof overhead and associated break-even financial points as affected bythe market price of livestock produced. The evaluation of the productioneconomics factors described above results in an economic productionmodel that is used to predict the value created by the technologysolution of the present invention taking into account the financialinvestment by the end user in the technology products of the presentinvention. The model predicts the effects on revenue and overallprofitability of the end-user on a per-crop and crop-to-crop basis andhelps to establish the economic framework on which the technologysolution is created for individual end users.

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views and moreparticularly to FIG. 1 thereof, there is shown an embodiment of thepresent invention.

The system of FIG. 1 includes a technology deployer 102, a manufacturer104, a distributor 106, end users 108 to 110 and field technicians/enduser advisors 112 to 114. It is noted that when those performing thefunction of 112 to 114, are the technical staff that work undercontract, or are employees or agents of the technology deployer 102and/or distributor 106, they can be referred to as Technology DeploymentManagers (TDMs). However, the function of 112 to 114 can be performed byfield technicians that are employees or agents of the end user 108 to110 and that have been appropriately trained by the technology deployer102. In FIG. 1, microbial samples 116 to 118 (e.g., pathogenic Vibrio )are collected by the field technicians 112 to 114 at the end userlocations 108 to 110. Physico-chemical data (e.g., dissolved oxygenconcentration, Ca concentration, pH, CO₂ concentration, salinity, etc.)are determined/analyzed (120 to 122) and transmitted (126 to 124) fromfield computers 128 to 130 to the technology deployer 102 computer 132.The determinations and analyses of the physico-chemical data from enduser 108 to 110 locations are used to generate technical recommendationsfor the end user 108 to 110 and these technical recommendations aretransmitted back (126 to 124) to the end user 108 to 110. Thesetechnical recommendations with regard to physical-chemical data can alsobe generated on site by the end user advisors 112 to 114. A laboratoryand computer 132 of the technology deployer 102 analyzes (134) themicrobial samples 116 to 118 to determine microbial strain compositionand/or quorum sensing molecule inhibitor identity to formulate productsand strategies to control and/or manage disease at the respective enduser locations 108 to 110 and transmits (148) the product designparameter and/or composition formulations to a computer 136 of themanufacturer 104. The determination of product design parameter and/orcomposition formulations include, for example, molecular and/orbiological analyses of samples 116 to 118 to identify probioticmicrobial bacterial strain(s) that can be used to help manage thedisease-causing biological samples 116 to 118 by producing enzymes,antimicrobial compounds, antibiotics, quorum sensing inhibitors andquorum sensing molecule enzyme degradation mechanisms that inhibit thegrowth and/or pathogenicity and thereby help manage disease related tothe samples 116 to 118 (e.g., as described in PCT Application SerialNumber PCT/US/16489 filed on 22 Jun. 2001). The manufacturer(s) 104 thenmanufactures the products consisting of microbial strain compositionsand/or quorum sensing inhibitor compounds 138 to 140 in concentratedform that then are sent to the distributor 106. A computer 142 at thedistributor 106 may also receive information 150 from the computer 132or information 154 from the computer 136 for further processing and/orcustom blending of the microbial strain compositions and/or quorumsensing inhibitor compounds 138 to 140 (e.g., carrier formulae,processing parameters, chemicals for spore activation, enrichment orsupplementation with specific microbial strains or chemicals indistributors inventory etc.), before being distributed as end user 108to 110 specific products 144 to 146. Thus, the distributor 106 may haveappropriate ingredients already in inventory to custom blend productsand/or further process products per information 150 provided from thecomputer 132 or per information 154 provided from computer 136.

The present invention thus includes training the distributors 106 andthe end users 108 to 110 to manage the microbial communities of aquaticfarms with the aid of the microbial biotechnology products includingprobiotics and/or quorum sensing inhibitor compound products 144 to 146and technical training services provided by the field technicians 112 to114 at the end user locations 108 to 110. The application of bio-controltechnology according to the present invention allows end users 108 to110, such as aquatic farmers, etc., to eliminate the use ofinappropriate or deleterious chemicals and antibiotics, which areharmful and largely ineffective. Once trained in the process ofbio-control of disease with technology according to the presentinvention, distributors 106 can expand their business replacing use ofharmful drugs and chemicals. The term bio-control as used herein refersto the use of beneficial, naturally occurring microorganisms and quorumsensing inhibitor compounds to prevent the infection and spread ofdisease in aquatic livestock.

According to the present invention, microbial technology productsinclude probiotics and/or quorum sensing inhibitor compounds 144 to 146,such as organic supplements and organic concentrates for aquaculture,and typically are sold in the form of dry powders and granules and arealso available in liquid form. These products are exported to thedistributors 106, who dilute and blend them according toinformation/guidelines 150 and/or 154 for forming fertilizers, feeds andother products enhanced with microbial technology as the principalactive ingredient. Fertilizers and feeds containing or carryingmicrobial technology are applied by the end users 108 to 110, such as ashrimp or fish producer, etc., directly at the end user locations, suchas, a pond, tank, etc., where the probiotic component of the microbialtechnology grow and flourish, fighting pathogenic bacteria and virusesin the water and in the gut of aquatic farm animals, preventinginfection and death.

According to the present invention, technical training services toenable the end user to develop and apply bio-control programs fully andachieve harvests and yields that typically are far superior to previousproductions are provided via field technicians/advisors 112 to 114. Forexample, end users 108 to 110, such as shrimp producers, fish producers,etc., that use the products 144 to 146 according to the presentinvention typically can eliminate the use of harmful chemicals and drugsin their animal production and prevent this route of entry into thehuman food chain. The products 144 to 146 according to the presentinvention include safe, environmentally friendly bacterial isolates fromnature, broadly classified as probiotics, and also quorum sensinginhibitor compounds that are natural products isolated originally frommarine algae [26] and are synthesized commercially. Because the products144 to 146 according to the present invention typically are notgenetically engineered, the products 144 to 146 typically do not haverestrictive regulatory requirements. Accordingly, the business potentialis excellent with large international markets ranging from Latin Americato Asia and where disease is a major threat to aquaculture.

The present invention could help the end users 108 to 110 combat complexdisease situations where environmental conditions are extreme anddifferent from those in other shrimp producing regions, for examplewhere the salinity is very high. According to the present invention,individual strains of microbes and/or quorum sensing inhibitor compounds138 to 140 that provide most protection against specific pathogens 116to 118 affecting the culture systems of the end user 108 to 110 areselected and new environment-specific products 144 to 146 are developed.In most shrimp producing countries the products 144 to 146 according tothe present invention may be sold to all major shrimp farms.

A focus of the present invention includes a strategy where products 144to 146 are continually developed against specific pathogens 116 to 118for the end user 108 to 110 working in specific environmental conditionsin addition to core products that provide protection broadly throughoutthe aquaculture industry. For example, if a new pathogenic strain ofVibrio were identified in a high salinity region, products 144 to 146can be developed according to the present invention to fight that strainof Vibrio specifically. The microbial technology products 144 to 146,developed according to the present invention, could provide control ofdisease in a very broad range of environments and under adverseconditions. According to the present invention an intensive R&D program134 is used to develop products 144 to 146 against new viral andbacterial pathogens.

According to the present invention, Technology Deployment Managers(TDMs) 112 to 114, trained and ready to support the end users 108 to 110and the distributors 106 are provided, while laboratory scientists,microbiologists and technical assistants (not shown) support productdevelopment work. As shown in FIG. 2, microbial technology products 202to 208 are developed and further processed 210 to 214, field tested andcommercialized into new commercial aquaculture end user markets 216 to220 including, for example, trout and salmon, tilapia, sea bass, seabream, snapper, eel, crab, turtle, scallops, oysters, pearl oysters,abalone, sea urchin, etc., are possible according to the presentinvention. The present invention further includes application ofproducts for disease control and water quality into the home andcommercial aquaria end user markets 222 and 224 including, for example,pet stores, fish and crustacean transport and holding facilities,restaurants, and high-density, closed fish production systems, etc. Thisapplication further includes microbes for bio-filters in such systems.FIG. 3 illustrates an exemplary device 304 in the form of a collectablecharacter for delivering microbes 302 in an aquarium 306 to help managethe microbial ecology of the aquarium 306 and thus prevent disease infish 308.

A business strategy according to the present invention is to combine themarket and technical strengths of the technology deployer 102 and withleast-cost production strengths of the manufacturers 104 to form aunique synergy, because of the facts that: (i) the manufacturers 104excel and focus in manufacturing bacteria or quorum sensing inhibitorcompounds, which are highly specialized processes, (ii) a manufacturer104 typically has strong capability in development of bacterial productssold to final product manufacturers 106 and (iii) the technologydeployer 102 has market and technical strengths not possessed by themanufacturer; and (iv) some manufacturers 104 typically have strongcapability in complex organic chemical synthesis. Thus, by combining thestrengths in manufacturing with applied the R&D, expert understandingand experience of the aquaculture market, technical innovation and salesand marketing strengths, such a combination has a strong advantage overcompetitors (e.g., example as described with respect to FIG. 10) thatare typically weak in manufacturing of bacteria and do not understandaquaculture markets or microbial ecology of aquaculture systems.

However, this is not to say that all critical functions ranging from R&Dto manufacturing to technology deployment to sales and marketing can notbe present in a single business entity that contains in all of theseskills, talents and functionalities among the operational units of asingle business entity formed for such a purpose.

According to FIG. 4, the business strategy according to an embodiment ofthe present invention takes advantage of least-cost manufacturing 104 ofhighly concentrated products 402, 412 that, for example, are: (i)further processed by a downstream manufacturer (404) into organicsupplements (406) for aquaculture and shipped to internationaldistributors (408) who further dilute or process the products into feeds(410) for animals or (ii) shipped directly to international distributors(414) as organic concentrates (412) for aquaculture and then are furtherprocessed (418) in-country. These strategies according to the presentinvention keep freight costs low by shipping low volumes of concentratedproducts (402, 412) directly into aquaculture markets and help keepaquaculture production costs low while managing disease.

According to the present invention, core market entry products are soldunder an umbrella trade mark and are comprised of two types: (i) thoseadded to aquaculture pond water (referred to as “aqua” products) and(ii) those added to the feed of aquatic animals (referred to as “feed”products). All products typically contain as an active ingredient, ablend of one or more species and strains of naturally occurring,benevolent microbes in a dry powder form and/or quorum sensing inhibitorcompounds. Products are also available in liquid form, as needed, forappropriate applications.

According to the present invention, there are, for example, two exportcategories: (i) organic supplements and (ii) organic concentrates. Theorganic supplements and organic concentrates can be standardized to anet weight with animal feed ingredients during further processing. Theabove-noted products are summarized in Table 1 below.

TABLE 1 Microbial Technology Products for Export Aqua Product 1—Anorganic supplement for aquaculture (aqua) including microbes and/orquorum sensing inhibitor compounds blended with animal feed qualitybyproducts, steam pelleted, crumbled and packaged into 5 to 50 kg Net Wtcontainers such as plastic bags in cardboard boxes. Aqua Product 2—Anorganic concentrate for aquaculture (aqua) including microbes,fermentation byproducts and single-cell protein products in dry powderor liquid form and/or quorum sensing inhibitor compounds and packaged inplastic-lined barrels, buckets or drums. Feed Product 1—An organicsupplement for aquaculture (feed) including microbes and/or quorumsensing inhibitor compounds blended with animal feed qualityby-products, steam pelleted, crumbled and packaged into 5 to 50 kg NetWt containers such as plastic bags contained in cardboard boxes. FeedProduct 2—An organic concentrate for aquaculture (feed) includingmicrobes, fermentation byproducts and single-cell protein products indry powder or liquid form and/or quorum sensing inhibitor compounds andpackaged in plastic-lined barrels, buckets or drums.

Another product according to the present invention is designed for theshrimp hatchery market and is described in Table 2 below. This productis used by end user consumers at low concentrations, added to tank waterand is high margin low-volume business. The export form is also theend-used form.

TABLE 2 Hatchery Products Hatchery Product—organic supplement foraquaculture (hatchery) including microbes, fermentation byproducts andsingle-cell protein products in dry powder or liquid form and/or quorumsensing inhibitor compounds, packaged in 100 gram to 50 kg containers.

The core aqua products for shrimp farms are further processed byblending with organic materials in country to form value-added microbialand/or quorum sensing inhibitor compound supplements carried as organicfertilizer products. Two concentrations, thus two local products asshown in Table 3 below, are sold locally depending on the method ofshrimp farming. The feed product is incorporated directly into the feedof aquatic animals by local feed manufacturers and thus requires nospecial handing by shrimp farmers.

TABLE 3 Microbial Technology Products for Local Distribution LocalProduct 1—Hi Concentration Aqua Product, an organic concentrate orsupplement for adding to aquaculture water (aqua) diluted at less thanor equal to about 1:1 to 1:5 with formulated carrier ingredients thatfunction as organic fertilizer for shrimp ponds. Local Product 2—LowConcentration Aqua Product, an organic concentrate or supplement foradding to aquaculture water (aqua) diluted at greater than or equal toabout 1:6 to 1:20 with formulated carrier ingredients that function asorganic fertilizer for shrimp ponds. Local Product 3—Feed, an organicconcentrate or supplement for aquaculture (feed) added to shrimp feed inthe range of about 0.1 to 100 kg per metric ton. Local Product4—Hatchery, microbes, fermentation byproducts and single-cell proteinproducts in dry powder or liquid form and/or quorum sensing inhibitorcompounds, packaged in 100 gram to 5 kg containers.

According to the present invention, the amount of product typicallyrequired by semi-intensive shrimp farmers is 0.1 to 100 kg per hectareper crop.

Training of the distributors 106 and end user 108 to 110 personnel is afeature of the present invention. Farmers need guidance and advice tomanage the microbial environment. Therefore, training services in theform of written technical bulletins, guidelines, and interactivemulti-media training modules such as through CD ROM or via the Internet,and with hands-on training in customer labs and in the field areprovided. The complete training program is focused on adult learningprinciples.

The immediate customers of the technology deployer 102 are thedistributors 106 of feed, chemicals and other products to the end users108 to 110, such as shrimp farmers, etc. The final end user customersare the aquaculture producers such as shrimp farmers 108 to 110.

Part of the uniqueness of the technology deployer 102, is that it is notsimply a production and sales company, but is at the forefront of itsscientific fields of microbial ecology and nutrition and brings newproducts based on new developments in microbial technology to themarketplace for the benefit of its customers.

Typically, all products containing microbes and/or quorum sensinginhibitor compounds are manufactured by the manufacturer 104 and mixedas dried powders or liquids in ratios appropriate to each final product(e.g., pond, feed, hatchery water, hatchery feed, etc., as previouslydescribed). The microbial and/or quorum sensing inhibitor products thenare shipped to the distributor 106 for further processing, finalblending, packaging and/or formulation as feed products. They are mixedwith feed materials supplied by other suppliers, and/or fortified orenriched with specific active ingredients, such as microbial strainsand/or quorum sensing inhibitor compounds, etc., and then packaged,labeled and shipped to final end user 108 to 110 destination or toanother location, depending on customer requirements. In eachdestination country, distributors 106 or end user farmers 108 to 110arrange for a feed manufacturer to incorporate the concentrate 144 intoa custom feed blend, containing about 0.1 to 100 kg concentrate/tonnefeed. Alternatively, end user customers 108 to 110 can buy a final feedmix 144 directly from the distributor 106.

The concentrated forms of the pond products may be formulated as crumblefeeds by the distributors 106. The concentrates for feed products (e.g.,for blending into shrimp feed) may be formulated as crumble feeds by thedistributors 106. The hatchery product concentrates (e.g., for hatcherytank water) may be formulated as powdered or liquid products by themanufacturer 104, then as a feed by the distributors 106.

The business of the technology deployer 102 typically is primarilycentered on applications of microbial and/or quorum sensing inhibitorcompounds technology, using products that are manufactured undercontract. Hence production typically is not a major facet of thetechnology deployer 102. The production of the microbial and/or quorumsensing inhibitor compound products 138 to 140 is of course complicated,which is why the technology deployer 102 contracts out the process tomanufacturers 104 with appropriate skills and facilities. However, allfacets of the applications of microbial and/or quorum sensing inhibitorcompounds technology and the production of the microbial and/or quorumsensing inhibitor compound products 138 to 140 may be performed by asingle business entity formed for such a purpose.

The microbial technology according to the present invention typically isa combination of products and know-how in applied microbial ecology,with the microbes occurring naturally in nature and without furthergenetic engineering. However, genetic engineering could be used inconjunction with the present invention, as will be appreciated by thoseskilled in the relevant art(s).

According to the present invention, techniques for controlling Vibriodisease using bioactive compounds to turn off genes for virulence (e.g.,as described in PCT Application Serial Number PCT/US/16489 filed on 22Jun. 2001) are also employed. Routine R&D is performed for improvingextant products and to select new microbial strains against Vibrio andvirus pathogens of shrimp.

According to the present invention, data are obtained for localconditions in each country as well as each region, as farmers are(justifiably) conservative and reluctant to accept studies done in otherregions before purchasing products. Using the methods described below,the research workers at the laboratory screen a range of potentialprobiotic bacterial species against pathogenic Vibrio strains isolatedfrom farms in each region and select probiotic strains that inhibit theVibrio species, using salt concentrations that are appropriate for theregion. For example, in some areas salinity may be as high as 6%, andthus probiotics must be selected that grow at 6% and are active againstVibrio or other pathogenic bacteria at 6% salinity.

New hatchery feeds and feed delivery methods that combine probioticmicrobes and bioactive compounds (e.g., as described in PCT ApplicationSerial Number PCT/US/16489 filed on 22 Jun. 2001) are part of thepresent invention, because feeds serve as important vehicles for diseasecontrol in the production of shrimp, fish, molluscs and other aquaticanimals in hatcheries, which are severely limited by such diseases.

According to the present invention, products for disease control areapplicable in many industries, not just aquaculture, and involvemolecular biotechnology, including bioactive compounds that turn offgenes for disease virulence in pathogens (e.g., as described in PCTApplication Serial Number PCT/US/16489 filed on 22 Jun. 2001). FIG. 5 isa diagram for illustrating bio-control of disease in water, according tothe present invention. In FIG. 5, bio-control of disease in water 502includes a comprehensive approach of monitoring/managing disease in thebiological community 504 using technology 512 to introduce microbialproducts 514 and/or bio-active compounds (e.g., quorum sensing inhibitorcompounds) 516 and to provide technical recommendations (124 to 126 ofFIG. 1) for managing the biological community 504, which includes, forexample, micro and macro algae 518 and 520, bacteria 522, which includespathogenic and beneficial bacteria 524 and 526, zooplankton 528,meiofauna 530, viruses 536, protozoans 538 and aquatic livestock 540.The bio-control of disease further includes controlling/collectingmicrobial factors 546 (116 to 118 of FIG. 1) and physico-chemicalfactors 506 and performing pond management 510, including providing feed542, via the technology 512 and the end user 508, as will be furtherdescribed in detail. Important cause and effect relationships areindicated in the drawing by arrows. The line drawn from viruses 536 topathogenic bacteria 524 represents control of pathogenic bacteria viainclusion of bacterial viruses as bioactive components according to thepresent invention.

FIG. 6 is a diagram for illustrating bio-control of disease in a gut ofan animal, according to the present invention. In FIG. 6, bio-control ofdisease 602 in a gut of an aquatic animal 604 includes a comprehensiveapproach of monitoring/managing disease in the gut of the aquatic animal604 using technology 512 introduced via the feed 542 including microbialproducts 514 and/or bio-active compounds (e.g., quorum sensing inhibitorcompounds) 516 and a nutrient formula 606 into the gut of the aquaticanimal 604, which can contain as a result of the animal's naturalforaging activity, for example, micro and macro algae 518 and 520,bacteria 522, which includes pathogenic and beneficial bacteria 524 and526, zooplankton 528, meiofauna 530, viruses 536, protozoans 538 andfeed 542. The bio-control of disease further includescontrolling/collecting microbial factors 546 (116 to 118 of FIG. 1) andphysico-chemical factors 506 and performing pond management 510, via thetechnology 512 and the end user 508, as will be further described indetail. Various cause and effect relationships are indicated in thedrawing by arrows.

FIG. 7 is a flow chart for illustrating the operation of the system ofFIG. 1 in controlling disease according to FIGS. 5 and 6, according tothe present invention. In FIG. 7, at step 702 the microbial samples 116to 118 are received from field technicians/TDMs 112 to 114 or directlyfrom other end user 108 to 110 staff and the physico-chemical data 124to 126 are received from the field computers 128 to 130 by thetechnology deployer computer 132. At step 704, the product designincluding the microbial strain composition and/or quorum sensinginhibitor compound formulation is determined/modified based on analysesof the received samples 116 to 118 and/or data 124 to 126 and a databaseof microbial strains and other product design parameters including feednutrient and feed nutrient formula 606, at the technology deployercomputer 132 and/or manufacturer computer 136 and/or distributorcomputer 142. The formulation may be transmitted by the technologydeployer computer 132 to the manufacturer computer 136 and/ordistributor computer 142 depending on the level of complexity orsimplicity of the product design modifications required to meet the enduser 108 to 110 requirements per analysis of samples 116 to 118 and/ordata 124 to 126 and a database of microbial strains and other productdesign parameters. For example, for a relatively simple modification toan existing product design, such a simple fortification or enrichmentrather than a complete product re-design, the technology deployer 102may transmit this information via the computer 132 or by other means ofcommunication to the manufacturer 104 or distributor 106 or directly tocomputers 136 and 142, respectively. In addition, at step 708, thetechnology deployer computer 132 may transmit to the end user 108 to 110end-user-specific technical management advise based on the receivedsamples 116 to 118 and/or data 124 to 126. Such data 124 to 126,formulation, pond management advice, etc., may also be transmitted viaany other suitable means, such as by facsimile, wireless communications,telephony, Internet, Intranet, modem, cellular phone, satellitecommunications, etc., as will be appreciated by those of ordinary skillin the relevant art(s).

At step 710, the manufacturer 104 manufactures the products 138 to 140based on the formulation. In addition, at step 710, the manufacturer 104or distributor 106 can mobilize from existing inventory the appropriatemicrobial strains or bioactive compound or other product components fora revised product design determined at step 704 and transmitted at step706 to meet revised product design criteria. The distributor 106 maythen further process the products for a given end user 108 to 110application at step 712. At step 714, the respective products 144 to 146are applied at the respective end user locations 108 to 110. The endusers 108 to 110 then monitor the respective end user locations. At step716, the respective end users 108 to 110 transmit updated microbialand/or physico-chemical samples 116 to 118 and data 124 to 126 to thetechnology deployer 102, completing the process.

The following sections describe the physico-chemical samples 116 to 118and data 124 to 126, which are collected and technical advice provided,which is followed by end user to obtain disease control, as describedwith respect to FIGS. 1-7. The examples below are described for shrimpaquaculture; for culture of other organisms, e.g. tilapia or salmon adifferent set of data would be obtained and analyzed and instructionsprovided for the end user. The examples provided above of biocontrol ofdisease in aquatic organisms takes into consideration the complexity ofraising animals in an aquatic environment but can be simplified andadapted for disease control in terrestrial farm animals per the presentinvention. For example, in the case of swine, poultry or cattleproduction the physico-chemical factors of the air and drinking waterare measured and analyzed and the biological factors associated with themicrobial flora of the feed and gut are considered.

Below is a list of physico-chemical and biological factors, theirmethods of analysis and optimum ranges for shrimp ponds (Table 4). Allend users are advised to determine this set of factors at the frequencylisted in the table. They record the information in a computerized formand transmit it by electronic means to the distributor and/or thetechnology deployer who monitor progress and provide advice where neededto bring the factors into the optimum range. The end user is advised ortrained where necessary with respect to how to manage the ponds to bringeach factor into its optimum range. For example, if calcium is low, thenthe end user will be advised to add lime; if oxygen is low, to add moreaerators or exchange water with filtered clean water (e.g., which may befrom a recycling reservoir, etc.).

TABLE 4 Factors to be determined in shrimp ponds. Optimum Value orFactors Method Frequency Range  1. Transparency Secchi disk daily, 12-1pm 35-50 cm  2. Oxygen DO/Temp meter daily, 5 am, 12-1 pm >4.0 ppm  3.Salinity Refractometer Weekly 20-35 ppt  4. Temperature DO/Temp meterdaily, 5 am, 12-1 pm 28-32° C.  5. Ph pH meter daily, 5 am, 12-1 pm8.0-8.3  6. Vibrio in water TCBS agar Weekly <1000-2000 cfu/ml  7.Luminous Vibrio - water TCBS agar Weekly <10 cfu/ml  8. Vibrio -hepatopancreas TCBS agar Weekly <1000/gut  9. Luminous V. hepatopancreasTCBS agar Weekly <10/gut 10. Alkalinity (units of CaCO₃) chemistry kitWeekly 120-130 ppm 11. Calcium hardness (as CaCO₃) chemistry kit Weekly250-400 ppm 12. Phosphate (before stocking) chemistry kit 1-2 days <0.8ppm 13. Phosphate (until week 4) chemistry kit Weekly <0.5 ppm 14.Ammonium (after week 12) chemistry kit Weekly <0.5 ppm 15. Nitrite(after week 12) chemistry kit Weekly <0.1 ppm 16. Vibrio in sedimentTCBS agar bi-weekly <2000 cfu/cc 17. Luminous V. sediment TCBS agarbi-weekly <10 /cc 18. Vibrio in haemolymph TCBS agar as desired 0 19.Algae: cell counts Microscope daily during >200,000/mL fertilizationMethods and Equipment

Kits with full instructions are available from several supply companiesdealing in water quality determinations. For a large farm or cooperativelaboratory, it is beneficial to have a chemistry and microbiologylaboratory with more elaborate equipment and trained staff.

Some factors vary diurnally and should be determined at the same time ofday for each sampling period.

Determination of Vibrio Bacterial Counts with Selective Media (e.g.,Note that Selective Microbiological Techniques May be Applied for otherPotential Bacterial Pathogens of Interest)

Use TCBS agar plates or dip slides. Dip Slides are plastic strips withTCBS agar in a sterile container. Dip slides can be taken to the pondsand dipped directly into the pond water. Incubate at 30-35° C. for 12hours, then count total luminous colonies. Prepare sterile dilutiontubes: (i) add 9 mL saline solution in 20-30 mL test tubes with loosecaps or cotton plugs; sterilize in autoclave and cool; (ii) water:collect samples of pond water in sterile bottles; (iii) sediment: seeabove section on sample collection; (iv) keep samples cool: 5-10° C.(refrigerator or ice pack) until ready to plate out. (v) homogenize 2 mLsamples.

Hepatopancreas Vibrio Counts (e.g., Note that Counts May also beDetermined for the Foregut and Haemolymph of Shrimp, as Needed)

Dissect the mid gut or hepatopancreas (mid gut gland) of the shrimp withclean scissors. Place the hepatopancreas in a clean dish. Using scissorscut the hepatopancreas up into small fragments, mix with 20-200 mLsterile saline water and homogenize. Plate out on TCBS agar usingstandard procedures or pour into clean Dip Slide container and dip theslide briefly, then incubate as described above.

Sediment Collection

Collect top 1 cm of sediment in PVC or Perspex tubes. Place sediment ina clean jar. Mix sediment with 50 mL filtered sterile water. Take 2 mlsubsample and homogenize then dilute 10-100 times for in filtered,sterile saline water. Plate out on TCBS agar using standard proceduresor pour into clean Dip Slide container and dip the slide briefly, thenincubate as described above.

Water Collection

Collect water near bottom in the ponds for analysis. In large ponds thatare not mixed, take several samples from around the pond and pool themto give an average sample.

Luminous Bacteria

The principal species that is luminous on TCBS is Vibrio harveyi,although not all strains of this species are luminous. There are otherluminous strains among other Vibrio genera, e.g. V. fisheri, V. choleraand V. splendidus. In general (but there are always exceptions), onlyluminous Vibrio luminesce on TCBS. The end user is advised to adjustdosing of the microbial technology products to keep luminous Vibrionumbers low or absent. If this cannot be achieved, then the end useradvises the distributor 106 and/or technology deployer 102 who willproceed to determine whether new or revised products are needed oradvise on varying application of current products.

The following section describes a summary of the shrimp pond samplingprocedures, and the type of advice given to the end users 108 to 110(e.g., shrimp farmers) to maintain water in optimum condition accordingto Table 4, so that added microbes can operate at optimum efficiency incontrolling pathogenic bacteria and viruses. By responding to adviceregarding these parameters, the end users 108 to 110 can maintain waterat a high enough quality to reduce the environmental stress on theaquatic livestock and help assure that the microbial products can workeffectively. As a consequence of making the physico-chemicaldeterminations, the end users 108 to 110 can be guided by the technologydeployer 102 according to the present invention in the most effectivetechnical management strategy.

Aeration to maintain dissolved oxygen concentration above 4 ppm andpreferably above 5 ppm, and mixing to maintain organic material insuspension with no deposition of organic sludge on the pond bottom, areimportant to help assist in maintenance of good water quality, which inturn allows added microbial products to work effectively.

Pond Preparation on Potential Acid Sulphate Soils

New Ponds: flush 4 to 5 times, preferably with fresh (non-saline) water.Between each flushing episode, allow pond to dry out and sedimentsbecome exposed to air for 2 to 3 days before flushing again. Monitor pHbefore and after flushing. When pH changes by less than 1 unit, drainthe pond, and apply hydrated lime (calcium hydroxide) at 1 tonne/hectareacross the soil. Then, apply agricultural lime (calcium carbonate) ordolomite at 2-5 tonne per hectare. Fill the pond with water and monitorpH. If it is less than pH 8.0, add more calcium carbonate. Betweencrops, the pond should be dried if possible, and black sludge removed.Monitor soil pH. If it less than pH 8.0, add 200-500 kg hydrated lime/haand 1-2 tonne/ha calcium carbonate or dolomite; the lower the pH, thelarger the dose of lime that should be added.

Products Added to the Aquafarm Pond Water

The hydration procedure for Aqua Product microbial technology is asfollows, but quantities and concentrations can be modified as needed:(i) use 1 kg per hectare per application; (ii) for each 1 kg of Aquaproduct, take 4 liters of fresh water If fresh water is not available,then use seawater that has been chlorinated for 24 hours with 100 ppmchlorine, then acidified, aerated and neutralized; (iii) add the Aquaproduct powder to a clean bin or other container that can be covered,then mix in the water; (iv) cover the bin and stand for 12 hours, thenmix with organic fertilizer or apply to feed; (v) hygiene is veryimportant, wash hands before preparing the Aqua Product suspension andwash all containers and mixing utensils before and after use.

Organic Fertilizer Medium for Aquatic Farm Ponds

The fertilizer medium is prepared as follows: (i) prepare hydrated Aquaproduct as described above 1 day before use; (ii) for each hectare,prepare a fermentation medium as follows: (a) yeast: 250 g, (b) soybeanmeal or meal flour: 25 kg, (c) rice bran: 25 kg, (d) fresh water 200liters, and (e) urea: 5 kg (see above: only if Secchi transparency is<60 cm) and soluble phosphate: 200 g (only if phosphate concentration inpond water is less than 0.1 ppm); (iii) mix together and add the 12 hourhydrated Aqua Product; and (iv) incubate for 2-3 days then spread overthe pond (if aeration is available, aerate during the incubationperiod).

Advice for Disease Control in Ponds With No Aeration.

At low stocking density typical of non-aerated ponds, applying diseasecontrol technology can be made more efficient by holding the postlarvae(seedstock) in an encierro, i.e., an enclosure comprised of nettingstretched across a pond or forming a cage within the larger pond, or ina nursery pond or raceway for 30 days after stocking or longer ifpossible. Animals should be concentrated at a high enough stockingdensity in the encierro enclosure to force them to eat artificial feedcontaining the microbial technology of the present invention. Beforeproceeding, farmers are advised and coached through a cost analysis inrelation to production objectives and variable such as stocking density,expected survival to determine the maximum encierro size consistent withcosts and expected financial returns for a given pond or set of ponds.

Encierros are built to have one or more phases or stages of enclosurefor progressively larger animals. For example, start with an encierro ofabout 500 to 700 square meters or of a size sufficient to hold newlystocked animals at a density of about 1000 to 5000 per tonne of water,then release animals into the second stage of 2500-3000 square meters orof a size sufficient to hold animals at a density of about 250 to 1000per tonne of water, then release animals into a larger encierro of about10,000-20,000 square meters pond area or of a size sufficient to holdanimals at a density of about 50 to 250 per tonne of water. Release theanimals from the smaller to larger encierros progressively, or transferanimals from smaller to larger containment systems (e.g., ponds, tanks)when growth starts to slow down, for example before oxygenconcentrations fall below 4 ppm and before the daily feeding ratereaches about 12 kg feed/ha/day. This will help maximize the amount oftime the animals are in water that is treated by the microbialtechnology of the present invention. An encierro can be made by placinga net of fine mesh (e.g., 0.5-1.0 mm) for smaller encierros or otherinexpensive barrier material around the water inlet or exit or fromdyke-to-dyke across the pond. The second net can be less expensive andmade of a larger mesh to hold larger animals. Feed trays should be usedat all times. It is best to apply as much feed as practicable to trays.Feed trays may be placed around the edge of the encierro for easyaccess, or to stakes across the pond or encierro.

The encierros should be treated with Hi Concentration Aqua Product atleast 2 days before stocking and then at 3-4 day intervals. In general,for newly stocked animals held at a density of about 1000 to 5000 pertonne of water, apply 1 kg of hydrated Hi Concentration Aqua Product per1,000 square meters of encierro surface; for animals held at a densityof about 250 to 1000 per tonne of water apply 0.5 kg (or more) ofhydrated Hi Concentration Aqua Product per 1,000 square meters ofencierro; for animals held at a density of about 50 to 250 per tonne ofwater apply 0.1 kg of hydrated Hi Concentration Aqua Product per 1,000square meters. The Low Concentrated Aqua Product is used for providing agreater organic fertilizer function in large-ponds systems whereencierros are not used and incoming water quality is low in nutrientsrequiring heavy fertilization.

If possible, use an aerator in the encierro; however, if this is notpractical, then water exchange should be used to keep dissolved oxygenabove 4 ppm. When a large exchange is made, add Aqua Product afterexchanging the water.

When growth rates slow down, the encierro should be expanded to a largerportion of the pond area or the juvenile shrimp should be released. AquaProduct may be applied to the whole pond 3 days before the juveniles arereleased from the encierro. Add 1 kg/ha of Hi Concentration Aqua Productor 10 kg/ha of Low Concentration Aqua Product to the whole pond 2-3 daysbefore releasing the juveniles, and as needed during the crop.

Response to Presence of Luminous Vibrio

A farmer may respond to presence of luminous Vibrio by managing thebacterial community composition to keep bacterial species balanced infavor of beneficial strains. The present invention provides a range ofspecially selected strains of microbial technology microbes that competewith and inhibit Vibrio in ponds and hatchery tanks and/or bioactivecompounds such as quorum sensing inhibitor compounds. When used with thetotal management system according to the present invention, includingphytoplankton management, Vibrio numbers typically can be controlled atless than about 1000 to 500/mL in intensive shrimp ponds. Some of thesemicrobial technology bacteria (Bacillus species) are selected for theircompetitive ability to remove the food of other heterotrophs such asVibrio , or their protective slime coats. Others are selected for theirdirect inhibitory effects on Vibrio harveyi (and certain otherpotentially pathogenic Vibrio species) through specific antibioticsecretion.

Algae can be controlled by manipulating nutrient ratios throughcontrolled feeding, and fertilizer additions, recycling of pond waterand using bacteria according to the present invention to compete withalgae for an essential nutrients. The following section describesHatchery Product application guidelines, according to the presentinvention.

Preparation of Hatchery Product Suspension

Prepare a suspension of 2 g/L for use in hatchery tank water and anotherof 100 g/L for mixing with hatchery feed: (i) heat 200 mL fresh water to65° C.; (ii) add the 2 or 100 g of the Hatchery. Product powder and mixwell; (iii) stand for 5 minutes; (iv) add 800 mL sterilized seawater atambient temperature (about 25° C.); (v) cover and stand for about 12hours at 30-35° C.; (vi) apply suspension to hatchery tank water dailyat about 500 ppm and to feed at 200 mL per kilogram; (vii) store extrasuspension for a further 12 hours maximum for mixing with feed; and(viii) store and refrigerate suspensions.

Application

Monitor Vibrio with TCBS medium in tanks and larvae. If numbers are toohigh, especially luminous Vibrio , double the dose of the HatcheryProduct suspension.

Feed Application:

Apply the Hatchery Product suspension (100 g/L) to feed for allbroodstock and larvae at 200 mL per kg feed by mixing suspension withfeed immediately before feeding. The feed may be stored chilled, but notfrozen, after mixing with the Hatchery Product suspension. The HatcheryProduct suspension should be mixed directly with all artificial feed forlarvae at 200 mL per kg feed by mixing suspension until it is absorbedby the feed immediately before feeding.

Artemia Tanks

Apply the Hatchery Product suspension (2 g/L) to Artemia cultures duringcyst hatching at 40 L per tonne of water and add daily to the Artemiatanks as needed. Check total and luminous Vibrio numbers at 24 hoursafter hatching; if Vibrio numbers are too high use increase doses.

Spawning Tanks:

Mix 500 mL Hatchery Product suspension (2 g/L) per 1 tonne (1000 L) ofwater in spawner holding tanks while eggs are released. Remove allfaeces of spawners from tanks.

Larval Rearing Tanks

N3 to Z3 stages: add 500 mL Hatchery Product suspension per tonne ofwater on the first day, then 250 mL until water exchange starts. Notethat the actual volumes and dosages may be varied from time to timedepending on product composition or varied in accordance with theparticular microbial community of a hatchery of the end user 108 to 110.

According to results of the microbial technology according to thepresent invention, in Panama, as described with respect to Table 5below, two farms, with both of the farms following guidelines and usingproducts according to the present invention in some or all of theirproduction ponds, reported good survival in the range of 60-90%survival, while many other farms in the country experienced heavymortality, 5-25% survival, due to White Spot Virus and pathogenicbacteria by day 30 to 40 of culture. In Table 5, the exemplary test datashowed a first profitable shrimp production in over 18 months in the twofarms in Panama, “La Fe” and “Anton” farms. The results are from a firstharvest during March-June 2000 in ponds 1-10 (La Fe) and P3 (Anton)using “best practice” recommendation with Aqua Product Encierro and FeedProduct according to the present invention. Profits were realized forthe first time in over 18 moths since White Spot Virus hit Panama in1999. Shrimp in ponds 1-10 and in P3 and P4 were from Panamanian stocks,which tested positive for White Spot Virus (by PCR, polymerase chainreaction test). Ponds 1-3 were Controls at La Fe originally using atreatment of brown sugar, which then were switched to the Aqua ProductEncierro and Aqua Product Feed according to the present invention atabout day 20 of culture and before any signs of disease were observed.Ponds P2 and P4 were brown sugar Controls, abandoned after diseasesymptoms appeared at about 45 days of culture. In Pond P1 the AquaProduct Encierro and Aqua Product Feed were not used in the manner usedin Pond P3 or ponds 1-10. During this trial most shrimp farmers inPanama continued to experience heavy mortalities by about 40 days ofculture with heavy financial losses to the industry. Thus these resultsshowed that when the microbial technology described in this inventionwas used, it was possible for farmers to produce crops successfully.

TABLE 5 Exemplary test data for Panama. Stock. Area Dens. SurvivalHarvest Profit Pond (ha) (per m²) (%) (kg/ha) ($/ha) Test Method  1^(a)1.2 10.1 92 1033 $4,181 Encierro; Panamanian larvae (seedstock)  2^(a)3.9 16.3 55 800 $2,501 Encierro; Panamanian larvae (seedstock)  3^(a)2.4 14.6 75 869 $1,955 Encierro; Panamanian larvae (seedstock)  4^(b)2.7 16.5 45 819 $2,024 Encierro Panamanian larvae (seedstock)  5 2.815.8 75 781 $1,186 Encierro; Panamanian larvae (seedstock)  6 2.7 15.486 1032 $2,260 Encierro Panamanian larvae (seedstock)  7 2.6 17.0 59 778$1,480 Encierro; Panamanian larvae (seedstock)  8 5.2 13.7 70 693 $1,685Encierro; Panamanian larvae (seedstock)  9 5.1 15.8 60 634 $1,015Encierro; Panamanian larvae (seedstock) 10 1.9 16.6 67 739 $641Encierro; Panamanian larvae (seedstock) P1^(c) 1.0 15.8 53 597 −$834Full Pond; SPF larvae (seedstock) P2^(d) 2.9 13.6 — — −$2,171 BrownSugar, abandoned day 45; SPF larvae (seedstock) P3 4.9 16.8 63 843$1,783 Encierro; Panamanian larvae (seedstock) P4^(d) 6.0 16.7 64 643−$623 Brown Sugar, abandoned day 45; Panamanian larvae (seedstock)^(a)CONTROL ponds treated with brown sugar switched to AquaProduct-Encierro and Aqua Product Feed by day 20, before experiencingdisease. ^(b)Harvest lasted 3 days due to drainage problem in pond: onthird day harvested 816 kg by manual cast net; survival artificially lowdue to drainage problem. ^(c)Aqua Product Encierro not used, rather AquaProduct Full Pond used with SPF seedstock. ^(d)CONTROL ponds treatedwith brown sugar switched to Aqua Product Encierro on day 45, afterexperiencing disease.

The following Table 6 contains results of commercial field trialsperformed between January and May 2001 in Machala Ecuador at shrimp farmBravo Grande of Pesquera Bravito. Three (3) commercial ponds (total of33.5 ha. stocked at 7.7 postlarval shrimp per m²) were used as controlswhere the standard technical management practices of the farm wereemployed; and three other test ponds (total of 31.5 ha. stocked at 7.5postlarval shrimp per m²) were treated with the technology of thepresent invention including use of the microbial products. As shown inTable 6, the ponds that were managed according to the biocontrol programof the present invention had significantly greater production and NetProfit relative to the control ponds.

TABLE 6 Exemplary test data for Ecuador. Factor Controls Test DifferenceProduction (lb/ha) 950 1230 280 Growth Rate 1.0 1.4 0.4 (g/week) FeedConversion 1.18 0.99 −0.19 Ratio Size at Harvest (g) 16.8 18.8 2.0 Daysof Culture 115 96 −19 Survival (%) 34 40 6% Product Expenses 0 $295 $295($/ha/crop) Net Profit ($/ha) $862 $1,855 $993

Table 7, shows results of the microbial technology according to thepresent invention, in hatcheries.

TABLE 7 Percent Survival of Larval L. vannamei grown in a commercialshrimp hatchery tanks during outbreak of the Zoea Syndrome. HATCHERYCONTROL PRODUCT 51 59 50 78 15 79 43 83 83 95 Average: 48.4 Average:78.8Analysis of Microbial Samples Taken from End User Locations

The present invention includes maintaining bacterial cultures on agarslopes, in frozen storage or as described in [21] or [22]. For screeningthe inhibitory effect of probiotic bacteria strains of the presentinvention on microbial samples taken from end user 108 to 110 locationswhere disease is present, a line is streaked across the center of apetri dish prepared with tryptone soy agar in 1%-6% NaCl. Incubation isperformed at 28-32° C. for 2 days, then the potential pathogenicbacterial strain (e.g., Vibrio strain to be tested) is cross streaked atright angles to the line of probiotic bacterial streaked previously(prepared as noted below). Incubation is then performed for a further 3days. Clear zones (i.e., no growth of Vibrio ) close to the probioticbacterial strain (e.g., Bacillus sp.) indicate inhibition of thepotential pathogen by the probiotic strain.

Pathogenic Vibrio strains: Grow these in nutrient broth in 2% NaClovernight, then transfer 0.1 mL to 10 mL of sterile 2% NaCl solutionbefore plating onto agar with Bacillus . Streak 5-10 μL with a loop.Tests for seeking and selecting probiotic bacteria active against otherpathogenic bacteria, whether marine or freshwater in origin, may beconducted in a similar manner.

FIG. 8 illustrates an overall network system diagram, according to thepresent invention. In FIG. 8, the system includes the field computers128 to 130, the technology deployment computer 132, the manufacturercomputer 136, the distributor computer 142 and respective databases 128a, 130 a, 132 a, 136 a and 142 a. The computers 128, 130, 132, 136 and142 are coupled via respective communications networks 802, 804, 806 and808, as shown in FIG. 8. The communications networks 802, 804, 806 and808 may be implemented via one or more communications networks (e.g.,the Internet, an Intranet and/or a combination of the Internet andIntranets) based on data security and other concerns, as will beappreciated by those skilled in the relevant art(s). In a preferredembodiment of the present invention, the communications network(s)preferably use electrical, electromagnetic, or optical signals thatcarry digital data streams.

It is to be understood that the system in FIG. 8 is for exemplarypurposes only, as many variations of the specific hardware used toimplement the present invention will be readily apparent to one havingordinary skill in the art. For example, the functionality of the variouscomputers 128, 130, 132, 136 and 142 may be implemented via one or moreprogrammed computers. To implement such variations as well as othervariations, a single computer (e.g., the computer system 901 of FIG. 9)may be programmed to perform the special purpose functions of thevarious computers shown in FIG. 8. On the other hand, two or moreprogrammed computers may be substituted for any one of the computers128, 130, 132, 136 and 142 shown in FIG. 8. Principles and advantages ofdistributed processing, such as redundancy and replication, may also beimplemented as desired to increase the robustness and performance of thesystem, for example.

Data structures are used to store the various data from theaforementioned discussions and include fields for storing same. Thepresent invention thus stores information relating to various processesdescribed herein. This information is stored in one or more memoriessuch as a hard disk, optical disk, magneto-optical disk, and/or RAM, forexample. One or more databases, such as the respective databases 128,130, 132, 136 and 142 shown in FIG. 8, may store the information used toimplement the present invention. The databases are organized using datastructures (e.g., records, tables, arrays, fields, graphs, trees, and/orlists) contained in one or more memories, such as the memories listedabove or any of the storage devices listed below in the discussion ofFIG. 9, for example.

All or a portion of the invention may be conveniently implemented usingconventional general purpose computers or microprocessors programmedaccording to the teachings of the present invention, as will be apparentto those skilled in the computer art. Appropriate software can bereadily prepared by programmers of ordinary skill based on the teachingsof the present disclosure, as will be apparent to those skilled in thesoftware art.

FIG. 9 illustrates a computer system 901 upon which an embodiment of thepresent invention may be implemented. Computer system 901 includes a bus903 or other communication mechanism for communicating information, anda processor 905 coupled with bus 903 for processing the information.Computer system 901 also includes a main memory 907, such as a randomaccess memory (RAM) or other dynamic storage device (e.g., dynamic RAM(DRAM), static RAM (SRAM), synchronous DRAM (SDRAM), flash RAM), coupledto bus 903 for storing information and instructions to be executed byprocessor 905. In addition, main memory 907 may be used for storingtemporary variables or other intermediate information during executionof instructions to be executed by processor 905. Computer system 901further includes a read only memory (ROM) 909 or other static storagedevice (e.g., programmable ROM (PROM), erasable PROM (EPROM), andelectrically erasable PROM (EEPROM)) coupled to bus 903 for storingstatic information and instructions for processor 905. A storage device911, such as a magnetic disk or optical disk, is provided and coupled tobus 903 for storing information and instructions.

The computer system 901 may also include special purpose logic devices(e.g., application specific integrated circuits (ASICs)) or configurablelogic devices (e.g., generic array of logic (GAL) or re-programmablefield programmable gate arrays (FPGAs)). Other removable media devices(e.g., a compact disc, a tape, and a removable magneto-optical media) orfixed, high-density media drives may be added to the computer system 901using an appropriate device bus (e.g., a small computer system interface(SCSI) bus, an enhanced integrated device electronics (IDE) bus, or anultra-direct memory access (DMA) bus). The computer system 901 mayadditionally include a compact disc reader, a compact disc reader-writerunit, or a compact disc jukebox, each of which may be connected to thesame device bus or another device bus.

Computer system 901 may be coupled via bus 903 to a display 913, such asa cathode ray tube (CRT), for displaying information to a computer user.The display 913 may be controlled by a display or graphics card. Thecomputer system includes input devices, such as a keyboard 915 and acursor control 917, for communicating information and command selectionsto processor 905. The cursor control 917, for example, is a mouse, atrackball, or cursor direction keys for communicating directioninformation and command selections to processor 905 and for controllingcursor movement on the display 913. In addition, a printer may provideprinted listings of the data structures/information or any other datastored and/or generated by the computer system 901.

The computer system 901 performs a portion or all of the processingsteps of the invention in response to processor 905 executing one ormore sequences of one or more instructions contained in a memory, suchas the main memory 907. Such instructions may be read into the mainmemory 907 from another computer readable medium, such as storage device911. One or more processors in a multi-processing arrangement may alsobe employed to execute the sequences of instructions contained in mainmemory 907. In alternative embodiments, hard-wired circuitry may be usedin place of or in combination with software instructions. Thus,embodiments are not limited to any specific combination of hardwarecircuitry and software.

As stated above, the system 901 includes at least one computer readablemedium or memory programmed according to the teachings of the inventionand for containing data structures, tables, records, or other datadescribed herein. Examples of computer readable media are compact discs,hard disks, floppy disks, tape, magneto-optical disks, PROMs (EPROM,EEPROM, Flash EPROM), DRAM, SRAM, SDRAM, etc. Stored on any one or on acombination of computer readable media, the present invention includessoftware for controlling the computer system 901, for driving a deviceor devices for implementing the invention, and for enabling the computersystem 901 to interact with a human user (e.g., a researcher, amanufacturer, a distributor, a filed technician, an end user, etc.).Such software may include, but is not limited to, device drivers,operating systems, development tools, and applications software. Suchcomputer readable media further includes the computer program product ofthe present invention for performing all or a portion (if processing isdistributed) of the processing performed in implementing the invention.

The computer code devices of the present invention may be anyinterpreted or executable code mechanism, including but not limited toscripts, interpreters, dynamic link libraries, Java classes, andcomplete executable programs. Moreover, parts of the processing of thepresent invention may be distributed for better performance,reliability, and/or cost.

The term “computer readable medium” as used herein refers to any mediumthat participates in providing instructions to processor 905 forexecution. A computer readable medium may take many forms, including butnot limited to, non-volatile media, volatile media, and transmissionmedia. Non-volatile media includes, for example, optical, magneticdisks, and magneto-optical disks, such as storage device 911. Volatilemedia includes dynamic memory, such as main memory 907. Transmissionmedia includes coaxial cables, copper wire and fiber optics, includingthe wires that comprise bus 903. Transmission media also may also takethe form of acoustic or light waves, such as those generated duringradio wave and infrared data communications.

Common forms of computer readable media include, for example, harddisks, floppy disks, tape, magneto-optical disks, PROMs (EPROM, EEPROM,Flash EPROM), DRAM, SRAM, SDRAM, or any other magnetic medium, compactdisks (e.g., CD-ROM), or any other optical medium, punch cards, papertape, or other physical medium with patterns of holes, a carrier wave(described below), or any other medium from which a computer can read.

Various forms of computer readable media may be involved in carrying outone or more sequences of one or more instructions to processor 905 forexecution. For example, the instructions may initially be carried on amagnetic disk of a remote computer. The remote computer can load theinstructions for implementing all or a portion of the present inventionremotely into a dynamic memory and send the instructions over atelephone line using a modem. A modem local to computer system 901 mayreceive the data on the telephone line and use an infrared transmitterto convert the data to an infrared signal. An infrared detector coupledto bus 903 can receive the data carried in the infrared signal and placethe data on bus 903. Bus 903 carries the data to main memory 907, fromwhich processor 905 retrieves and executes the instructions. Theinstructions received by main memory 907 may optionally be stored onstorage device 911 either before or after execution by processor 905.

Computer system 901 also includes a communication interface 919 coupledto bus 903. Communication interface 919 provides a two-way datacommunication coupling to a network link 921 that may be connected to,for example, a local network 923. For example, communication interface919 may be a network interface card to attach to any packet switchedlocal area network (LAN). As another example, communication interface919 may be an asymmetrical digital subscriber line (ADSL) card, anintegrated services digital network (ISDN) card or a modem to provide adata communication connection to a corresponding type of telephone line.Wireless links may also be implemented. In any such implementation,communication interface 919 sends and receives electrical,electromagnetic or optical signals that carry digital data streamsrepresenting various types of information.

Network link 921 typically provides data communication through one ormore networks to other data devices. For example, network link 921 mayprovide a connection to a computer 925 through local network 923 (e.g.,a LAN) or through equipment operated by a service provider, whichprovides communication services through a communications network 927. Inpreferred embodiments, local network 923 and communications network 927preferably use electrical, electromagnetic, or optical signals thatcarry digital data streams. The signals through the various networks andthe signals on network link 921 and through communication interface 919,which carry the digital data to and from computer system 901, areexemplary forms of carrier waves transporting the information. Computersystem 901 can transmit notifications and receive data, includingprogram code, through the network(s), network link 921 and communicationinterface 919.

Recapitulating, with multiple pathogens now present in the environment:pathogenic Vibrios, White Spot Virus, Taura Syndrome Virus, Yellow HeadVirus, NHP and others—the methods of aquaculture have changed forever.In this respect, the present invention includes recognition that simplyadding beneficial bacteria to aquaculture systems typically does notnecessarily provide a solution to disease. The addition of bacteria mustbe integrated into a wider application of principles of microbialecology [10, 12-14].

The present invention further includes recognition that pond managementadvice, as related to economics of production, is affected by diseaseand level of biocontrol designed for the end user: For example, iffarmer A wants 95% survival of 20 gram shrimp harvested at 6000 kg perhectare, costs will be much higher, as will yields, and a very differentbiocontrol program will be designed than for a farmer B who wants 750kg/ha of 14 gram shrimp and can do well for his goals with 45% survival.In this case, farmer A can spend as much as $1200/ha on biocontrol,while farmer B can spend as much as $350/ha on biocontrol.

In the above respect, the present invention provides a uniquecombination of technology, experience, reputation and know-how that helpto control disease in aquaculture by adapting to the new set ofconditions resulting from the way microbes are evolving.

Although the present invention is described in terms of disease controlfor aquaculture applications, food processing applications, commercialaquaria applications, hobby aquaria applications, etc., the presentinvention may be practiced to control disease in other applications,such as in terrestrial farm animal production, etc., as will beappreciated by those skilled in the relevant art(s).

Although the present invention is described in terms of gatheringsamples and/or data using laboratory techniques, the present inventionmay be practiced by collecting samples and/or data using bio-chips andbio-sensors as taught in, for example, [23-25], as will be appreciatedby those skilled in the relevant art(s).

Numerous modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

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1. A process for controlling disease in aquatic animals at an end userlocation, comprising: (a) testing a range of candidates including atleast one of: (i) Bacillus species, (ii) Bacillus strains, (iii) speciesof beneficial bacteria (iv) strains of beneficial bacteria and (v)strains of beneficial bacterial viruses, against samples including atleast one of pathogenic Vibrio,Gram negative pathogenic bacteria andGram positive pathogenic bacteria taken from an end user location;performing the step of selecting one or more of the candidates thatinhibit and attack at least one of the samples by at least one of insitu antibiotic production, competitive exclusion, and production ofenzymes that degrade quorum sensing molecules, (b) testing a range ofquorum sensing inhibitor compounds against the samples; and (c)performing the steps (a) and (b) for the end user location, includingone of a country, major region and individual end user location, todetermine microbial strain composition based on the selected candidateand/or quorum sensing inhibitor compounds and the tested range of quorumsensing inhibitor compounds to formulate a product and target microbialtechnology for use in bio-control of disease specific to the end userlocation.
 2. The process of claim 1, further comprising: receiving atleast one of physico-chemical data, biological data and management datafrom the end user location; and providing at least one of a generalbio-control program, a customized bio-control program, a generalbio-control product and a customized bio-control product to managedisease at the end user location based on at least one of the receivedphysicochemical data, biological data and management data and thesamples.
 3. The process of claim 2, wherein the step of providinggeneral and customized bio-control programs includes providing at leastone of pond management advice, pond bottom preparation advice,fertilization advice, stocking advice, water management advice, aerationadvice, nutrition advice, feeding advice, hatchery culture advice,grow-out production advice, advise on the application and dose rates ofmicrobial technology products to water and feed, training services andmanagement advice as related to economics of production, specific to theend user location.
 4. A computer-readable medium carrying one or moresequences of one or more instructions for controlling disease in aquaticanimals at an end user location, the one or more sequences of one ormore instructions including instructions which, when executed by one ormore processors, cause the one or more processors to perform the stepsof: (a) testing a range of candidates including at least one of: (i)Bacillus species, (ii) Bacillus strains, (iii) species of beneficialbacteria (iv) strains of beneficial bacteria and (v) strains ofbeneficial bacterial viruses, against samples including at least one ofpathogenic Vibrio, Gram negative pathogenic bacteria and Gram positivepathogenic bacteria taken from an end user location; performing the stepof selecting one or more of the candidates that inhibit and attack atleast one of the samples by at least one of in situ antibioticproduction, competitive exclusion, and production of enzymes thatdegrade quorum sensing molecules, (b) testing a range of quorum sensinginhibitor compounds against the samples; and (c) performing the steps(a) and (b) for the end user location, including one of a country, majorregion and individual end user location, to determine microbial straincomposition based on the selected candidate and/or quorum sensinginhibitor compounds and the tested range of quorum sensing inhibitorcompounds to formulate a product and target microbial technology for usein bio-control of disease specific to the end user location.
 5. Thecomputer-readable medium of claim 4, further comprising: receiving atleast one of physicochemical data, biological data and management datafrom the end user location; and providing at least one of a generalbio-control program, a customized bio-control program, a generalbio-control product and a customized bio-control product to managedisease at the end user location based on at least one of the receivedphysicochemical data, biological data and management data and thesamples.
 6. The computer-readable medium of claim 5, wherein the step ofproviding general and customized bio-control programs includes providingat least one of pond management advice, pond bottom preparation advice,fertilization advice, stocking advice, water management advice, aerationadvice, nutrition advice, feeding advice, hatchery culture advice,grow-out production advice, advise on application and dose rates ofmicrobial technology products to water and feed, training services andmanagement advice as related to economics of production, specific to theend user location.
 7. A system for controlling disease in aquaticanimals at an end user location, comprising: (a) means for testing arange of candidates including at least one of: (i) Bacillus species,(ii) Bacillus strains, (iii) species of beneficial bacteria (iv) strainsof beneficial bacteria and (v) strains of beneficial bacterial viruses,against samples including at least one of pathogenic Vibrio, Gramnegative pathogenic bacteria and Gram positive pathogenic bacteria takenfrom an end user location; means for selecting one or more of thecandidates that inhibit and attack at least one of the samples by atleast one of in situ antibiotic production, competitive exclusion, andproduction of enzymes that degrade quorum sensing molecules, (b) meansfor testing a range of quorum sensing inhibitor compounds against thesamples; and (c) means for performing (a) and (b) for the end userlocation, including one of a country, major region and individual enduser location, to determine microbial strain composition based on theselected candidate and/or quorum sensing inhibitor compounds and thetested range of quorum sensing inhibitor compounds to formulate aproduct and target microbial technology for use in bio-control ofdisease specific to the end user location.
 8. The system of claim 7,further comprising: means for receiving at least one of physicochemicaldata, biological data and management data from the end user location;and means for providing at least one of a general bio-control program, acustomized bio-control program, a general bio-control product and acustomized bio-control product to manage disease at the end userlocation based on at least one of the received physicochemical data,biological data and management data and the samples.
 9. The system ofclaim 8, wherein the means for providing general and customizedbio-control programs includes means for providing at least one of pondmanagement advice, pond bottom preparation advice, fertilization advice,stocking advice, water management advice, aeration advice, nutritionadvice, feeding advice, hatchery culture advice, grow-out productionadvice, advise on the application and dose rates of microbial technologyproducts to water and feed, training services and management advice asrelated to economics of production, specific to the end user location.10. A system configured to control disease in aquatic animals at an enduser location, comprising: (a) a technology deployer configured to testa range of candidates including at least one of: (i) Bacillus species,(ii) Bacillus strains, (iii) species of beneficial bacteria (iv) strainsof beneficial bacteria and (v) strains of beneficial bacterial viruses,against samples including at least one of pathogenic Vibrio, Gramnegative pathogenic bacteria and Gram positive pathogenic bacteria takenfrom an end user location; the technology deployer further configured toperform the step of selecting one or more of the candidates that inhibitand attack at least one of the samples by direct inhibition of at leastone of in situ antibiotic production, competitive exclusion, andproduction of enzymes that degrade quorum sensing molecules, and (b)testing a range of quorum sensing inhibitor compounds against thesamples, wherein the technology deployer is further configured toperform (a) and (b) at the end user location, including one of acountry, major region and individual end user location, to determinemicrobial strain composition based on the selected candidate and/orquorum sensing inhibitor compounds and the tested range of quorumsensing inhibitor compounds to formulate a product and target microbialtechnology for use in bio-control of disease specific to the end userlocation.
 11. The system of claim 10, wherein the technology deployer isfurther configured to receive at least one of physicochemical data,biological data and management data from the end user location; and thetechnology deployer is further configured to provide at least one of ageneral bio-control program, a customized bio-control program, a generalbio-control product and a customized bio-control product to managedisease at the end user location based on at least one of the receivedphysicochemical data, biological data and management data and thesamples.
 12. The system of claim 11, wherein the general and customizedbio-control programs include at least one of pond management advice,pond bottom preparation advice, fertilization advice, stocking advice,water management advice, aeration advice, nutrition advice, feedingadvice, hatchery culture advice, grow-out production advice, advise onthe application and dose rates of microbial technology products to waterand feed, training services and management advice as related toeconomics of production, specific to the end user location.